The present invention is an automated system for preparing and delivering sample mixtures to a chemical analyzer capable of accommodating liquid sample introduction, as well as washing the chemical analyzer.
Atomic absorption spectroscopy (AAS) is a common, well known technique for elemental chemical analysis. The most common AAS apparatus uses a flame as a means of atomizing the sample. This apparatus setup is known as flame AAS, or FAAS. Typically, sample is introduced into the flame by means of a nebulizer. FAAS typically needs at least a 2-10 mL volume of sample in order to run an analysis.
The xe2x80x9cthroughputxe2x80x9d of an analytical technique is a performance characteristic and is determined primarily on how many samples can be analyzed in a given period of time. The throughput of FAAS is generally poor, because there are requirements of sample preparation and apparatus cleaning that go along with each sample analysis. For example, the sample to be analyzed is often mixed with other agents (suppressors, matrix modifiers, releasing agents, etc.) before it is introduced for analysis. In addition, the nebulizer system must be washed between successive sample introductions in order to avoid memory effects due to remnants of previously analyzed samples.
Some FAAS systems are equipped with autosampler systems. These autosamplers can be programmed by the operator to analyze samples without operator intervention. The samples to be analyzed must be located in specialized sample trays whose locations are known by the autosampler. Such automated devices do reduce the amount of time and effort required by the operator for analysis of samples, but they do not improve the throughput performance of the instrument since the requirements of sample preparation and washing still exist. Therefore, there is a need for an analysis method that not only automates sample analysis, but also automates washing, and sample preparation.
In terms of the application of FAAS to pharmaceutical and biomedical research, samples from drug discoveries are often as small as 10-200 xcexcL, and are therefore very difficult, if not impossible, to analyze by FAAS using direct sampling from a microplate. In most cases, these small volume samples require dilution to bring the sample into a useful range, which dilutes the analyte concentration and sacrifices sensitivity. Ion channel assays (e.g. determinations of potassium, calcium, sodium, chloride, or rubidium concentrations in the ion channels) are subject to this same kind of limitation. As such, there is a need for a sample analysis system which allows small sample sizes (e.g. in the order of 10-200 xcexcL) to be analyzed without dilution.
Traditionally, analytical applications for ion channel analysis have fallen on either of the extremes of accuracy or speed. Presently ion channel assays are not fully automated to maximize sample throughput. The patch-clamp method is indisputably the most accurate, but it has a low throughput speed. Fluorescent dye measurements offer unsurpassed analysis speed, but suffer from low accuracy. Furthermore, other techniques that manage to sit in the middle ground between high accuracy and fast speed do possess equally limited disadvantages. The radioactive 86Rb+ efflux assay, for example, is a relatively unsafe and inconvenient technique in that the radioactive isotopes required are harmful to human operators, the half-life of the isotopes restricts the time duration of experiments, and there are radioactive waste disposal considerations to be dealt with.
Recently, Georg C. Terstappen described a method of using FAAS for an ion channel assay of rubidium. His method involved the dilution of the original sample 10-25 fold with an ion suppressor in order to obtain a sample volume that could be analyzed with FAAS. Such a dilution results in a significant loss in sensitivity for the analysis (10-25 fold). In addition, the dilution was performed manually, not automatically, so the sample throughput of the technique was significantly decreased. Therefore, there is a need in the analytical instrumentation industry to develop innovative analysis solutions for ion channel assays.
For example, potassium ion (K+) channels are critical aspects of many cellular processes within the human body. K+channel modulators offer significant therapeutic solutions to a variety of pathophysiological conditions. Therefore, innovations in the evaluation of K+channel activity would greatly support both academic and pharmaceutical research in this area. As such, there is a need for a method to quickly, easily, and accurately analyze ion channel assays, as well as pharmaceutical drug candidates, such as for ones that block the hERG K+ channel (which are vital for regulating the balance of pro- and anti-arrhythmic potentials) and prolong the QT interval.
As a result it is an object of this invention to provide a safe and reliable compromise between the traditional one-sided extremes of speed and accuracy associated with ion channel assay analysis. It is a further object of this invention to provide an analysis method that not only automates sample analysis, but also automates washing, and sample preparation, such that typical low sample throughput of most instruments, such as FAAS, is improved. It is still a further object of this invention to provide an analysis method that enables microsampling without dilution, such that minimum sample sizes for FAAS analysis can be lowered.
The present invention is a fully automated chemical analysis system and is particularly well suited for pharmaceutical drug discovery and biomedical research applications (for example, ion channel assays), compromised in part by an electronically controlled microsyringe pump, an injection port, a nebulizer and a FAAS instrument. The different components of the system are connected by tubing, allowing solutions to be exchanged between the various components of the system. The chemical analysis system further includes an autosampler, such as the XYZ autosampler available from Aurora Instruments, an array of sample microplates, and an array of solution containers (for standards, modifiers, buffers, suppressors, etc.). The chemical analysis instrument used in this invention can be any one of a multitude available on the market today, as long as it can accommodate liquid sample introduction. The FAAS instrument, however, will likely be the most useful chemical analysis instrument for many applications.
One advantage of the present invention is that it allows for a direct injection of small volumes of samples (in the order of 10-200 xcexcL) into the nebulizer of a FAAS. As the invention permits the analysis of microliter sample volumes, the need to dilute samples and sacrifice sensitivity is avoided. The use of the electronically controlled microsyringe pump allows for accurate analysis of such small sampling volumes.
In contrast to the prior art, the use of electronically controlled microsyringe pump and autosampler enables the time-consuming task of aspirating, mixing, and dispensing sample mixtures, as well as washing the sampling apparatus to be performed in a single sampling step, resulting in an increased throughout. The throughput of the apparatus may be further increased by the employment of multiple sample channels simultaneously. As a result, a further advantage of the present invention is that it can incorporate any number of parallel chemical analysis instrument channels (from one up to as many as is practically achievable).
A further advantage of the present invention is the ability of the apparatus to perform auto-dilutions of solutions and calibrations from a single standard. The full automation of auto-dilutions and calibrations relieves the human operator of the time and effort normally required of conventional chemical analysis systems on the market.
Still, a further advantage of the present invention is that the washing aspect of that sampling step performed by the microsyringe pump and injection port is very effective at reducing memory efforts (contamination problems between successive samples due to residues of past samples left in the instrument). Memory effects are a common concern with most automated chemical analysis systems.